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Volume 110 (1991)
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- Issue 3 Pages 315-
- Issue 2 Pages 163-
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Volume 110, Issue 3
Displaying 1-27 of 27 articles from this issue
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Akio Kanatani, Toyofumi Masuda, Taiji Shimoda, Fusakazu Misoka, Xu She ...1991 Volume 110 Issue 3 Pages 315-320
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProtease II gene of Escherichia coli HB101 was cloned and expressed in E. coli JM83. The transformant harboring a hybrid plasmid, pPROII-12, with a 2.4 kbp fragment showed 90-fold higher enzyme activity than the host. The whole nucleotide sequence of the inserted fragment of plasmid pPROII-12 was clarified by the dideoxy chain-terminating method. The sequence that encoded the mature enzyme protein was found to start at an ATG codon, as judged by comparison with amino terminal protein sequencing. The molecular weight of the enzyme was estimated to be 81, 858 from the nucleotide sequence. The reactive serine residue of protease II was identified as Ser-532 with tritium DFP. The sequence around the serine residue is coincident with the common sequence of Gly-X-Ser-X-Gly, which has been found in the active site of serine proteases. Except for this region, protease II showed no significant sequence homology with E. coli serine proteases, protease IV and protease La (lon gene), or other known families of serine proteases. However, 25.3% homology was observed between protease II and prolyl endopeptidase from porcine brain. Although the substrate specificities of these two enzymes are quite different, it seems possible to classify protease H as a member of the prolyl endopeptidase family from the structural point of view.View full abstractDownload PDF (2498K) -
Daita Nadano, Toshihiro Yasuda, Koichiro Kishi1991 Volume 110 Issue 3 Pages 321-323
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDeoxyribonuclease I (DNase I) was purified about 850, 000-fold from human kidney using a rabbit anti-human urine DNase I antibody and sensitive DNase I activity assay. On SDS-PAGE, the purified kidney DNase I gave a single major band, and its molecular mass was estimated to be 38, 000 Da. The activity of purified kidney DNase I was dependent on the presence of Mg2+ and Ca2+. G-Actin inhibited the activity, as did the anti-urine DNase I antibody. The properties of the kidney DNase I were the same as those of urine DNase I.View full abstractDownload PDF (314K) -
Sumio Maeda, Katsuhiko Takayanagi, Yoshifumi Nishimura, Takemi Maruyam ...1991 Volume 110 Issue 3 Pages 324-327
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSExpression of the Escherichia coli outer membrane proteins, OmpC and OmpF, is regulated in response to the medium osmolarity. The OmpR and EnvZ proteins are transcriptional factors involved in this osmotic regulation of the ompC and ompF genes. In particular, expression of the ompC gene is activated by the positive regulator, OmpR, in response to high osmolarity of the medium. In this study, we succeeded in defining a functional OmpR-binding sequence by analyzing a set of synthetic oligonucleotides, and propose a consensus motif for OmpR-binding. It was also demonstrated that the asymmetric OmpR-binding sequence, thus identified, can activate the canonical ompC promoter in an orienta-tion independent-manner, providing that this sequence is placed closely and stereospecifically with respect to the -35 region.View full abstractDownload PDF (834K) -
Makoto Ito, Yuko Ikegami, Akira Omori, Tatsuya Yamagata1991 Volume 110 Issue 3 Pages 328-332
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEndoglycoceramidase (EGCase) cleaves the linkage between oligosaccharides and ceramides of various glycosphingolipids [Ito, M. & Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282]. A detergent was required for EGCase to express full activity, possibly due to its hydrophobic nature. Recently, activator proteins responsible for stimulating EGCase activity in the absence of detergents were isolated from the culture supernatant of Rhodococcus sp. [Ito, M., Ikegami, Y., & Yamagata, T. (1991) J. Biol. Chem. 266, 7919-7926]. The activity of activator II specific for EGCase II was heat-labile but insensitive to trypsin-treatment. This activator (69.2 kDa) was converted to the 27.9 kDa polypeptide via the 42 kDa intermediate by exhaustive trypsination, and the stimulatory activity of 27.9kDa polypeptide on EGCase II was identical to that of the native form toward asialo GM1 and cell-surface GM3 of horse erythrocytes as substrates. This observation was successfully applied to obtain the purified activator without contamination with EGCase activity, which is abolished completely following treatment with trypsin.View full abstractDownload PDF (570K) -
Tamiko Suzuki-Nishimura, Masaatsu K. Uchida1991 Volume 110 Issue 3 Pages 333-338
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn the presence of bovine brain calmodulin activated by calcium, the sharp triplet electron spin resonance (ESR) lines of free doxyl stearic acids decreased, and the broad resonance lines increased concomitantly, suggesting that the doxyl stearic acids bound to calmodulin calcium-dependently. The bound molecules were displaced by a calmodulin inhibitor, W-7, whereas their nitroxide radicals were hardly reduced by ascorbic acid, suggesting that the spin-labeled fatty acids bind to hydrophobic regions of calmodulin, and consequently inhibit calmodulin-dependent phosphodiesterase activity. These binding characteristics to calmodulin were different from those to bovine serum albumin. Moreover, the ESR spectra of two spin-labeled derivatives of lysophospholipid having a spin-labeled acyl group or a spin-labeled polar head group showed that it is the acyl chain of lysophospholipid that interacts with the hydrophobic region of calmodulin. The interactions of fatty acids and lysophospholipids with calmodulin seem to be quite different from those of acidic phos-pholipids, described previously [ Suzuki, T., Katoh, H., & Uchida, M. K. (1986) Biochim. Biophys. Acta, 873, 379-386]. Thus, from the results of ESR study, we can obtain information on the function of fatty acids and lysophospholipids on calmodulin. Instead of enzyme assay, ESR spectroscopy is a useful means to examine lipid-protein interaction.View full abstractDownload PDF (647K) -
Hideaki Tsuyuki, Kenta Kajiwara, Akira Fujita, Takashi Kumazaki, Shin- ...1991 Volume 110 Issue 3 Pages 339-344
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo metalloendopeptidases, designated as Streptomyces griseus metalloendopeptidases I and II (SGMPI and SGMPII), were isolated from a commercial Pronase P by a method including affinity chromatography on carbobenzoxy-L-alaninyl-triethylenetetraminyl-Sepharose (Z-Ala-T-Sepharose). The two enzymes differed from each other in behavior on ion-exchange chromatography but showed the same amino-terminal sequence at least up to the 20th residue. Their molecular weights were both estimated to be 37, 000 by SDS-polyacrylamide gel electrophoresis. Elemental and amino acid composition analyses indicated that both of them contained about 1 g atom of zinc and one cystine residue per mol of protein. Cleavage specificities of the two enzymes toward synthetic peptide-substrates were very similar to those observed with thermolysin. EDTA, o-phenanthroline, and phosphoramidon strongly inhibited these enzymes, while typical serine-protease in-hibitors and cysteine-protease inhibitors had no effect. The findings clearly indicate that SGMPI and SGMPII can be classified into the family of zinc-endopeptidases. It was unexpectedly found, however, that these metalloendopeptidases were strongly inhibited by protein serine-protease inhibitors produced by Streptomycetes, such as Streptomyces subtilisin inhibitor (SSI), alkaline protease inhibitor-2c' (API-2c'), and plasminostreptin (PS).View full abstractDownload PDF (1921K) -
Kenta Kajiwara, Takashi Kumazaki, Eisuke Sato, Yuichi Kanaoka, Shin-ic ...1991 Volume 110 Issue 3 Pages 345-349
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA spectrofluorometric method for sensitive determination of metalloendopeptidase activity has been developed by using a bimane-peptide containing a tryptophan residue, i. e. 1, 7-dioxo-2, 5, 6-trimethyl-1H, 7H-pyrazolo [1, 2-a] pyrazol-3-yl-methyl-thiomethylcarbonyl-phenylalanyl-tryptophanyl-leucine (Bim-SCH2CO-Phe-Trp-Leu-OH). Such an “intramo-lecularly quenched” substrate was originally designed for a sensitive assay of angiotensin I converting enzyme (ACE) [ Sato, E. et al. (1989) Chem. Pharm. Bull. 37, 145-147]. All the typical metalloendopeptidases tested, such as thermolysin, Pseudomonas aeruginosa (Ps.) elastase, Streptomyces griseus metalloendopeptidases I and II (SGMPI and SGMPII), and alkinonase A, a metalloendopeptidase from Streptomyces violaceorectus, cleaved this substrate strictly at a Phe-Trp bond, leading to a marked increase in fluorescence. Kinetic parameters of the enzymatic hydrolyses of five kinds of analogous bimane substrates were compared to examine how the nature of neighboring amino acid residues on either side of the cleavable bond affects the catalytic efficiency of each of the metalloendopeptidases. Bim-SCH2CO-Phe-Trp-Leu-OH was most efficiently hydrolyzed by all of these enzymes. The use of this substrate made it possible to determine minute amounts of metalloendo-peptidases, especially those originating from Streptomycetes (for example, as little as 10 fmol of SGMPII).View full abstractDownload PDF (529K) -
Kenta Kajiwara, Akira Fujita, Hideaki Tsuyuki, Takashi Kumazaki, Shin- ...1991 Volume 110 Issue 3 Pages 350-354
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStreptomyces griseus metalloendopeptidase II (SGMPII) was shown to form tight complex-es with several Streptomyces protein inhibitors which had been believed to be specific to serine proteases, such as Streptomyces subtilisin inhibitor (SSI), plasminostreptin (PS), and alkaline protease inhibitor-2c' (API-2c'), as well as with Streptomyces metalloprotease inhibitor (SMPI). The dissociation constants of complexes between SGMPII and these inhibitors were successfully determined by using a novel fluorogenic bimane-peptide substrate. The values ranged from nM to pM. The results of studies by gel chromatographic and enzymatic analyses indicated that SGMPII is liberated from the complex with SSI by the addition of subtilisin BPN'. SGMPII and subtilisin BPN' proved, therefore, to interact with SSI in a competitive manner, despite the difference in the chemical nature of their active sites.View full abstractDownload PDF (644K) -
Takeo Yamaguchi, Takuya Kajikawa, Eiji Kimoto1991 Volume 110 Issue 3 Pages 355-359
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen human erythrocytes were subjected to hydrostatic pressure (1.1-2.0 kbar), it was found that membrane vesicles were released from the red cells above 1. 4 kbar. As with hemolysis under high pressure, the amount of released vesicles was increased with increasing pressure but decreased by the cross-linking of membrane proteins with diamide. Vesicles obtained at 2.0 kbar were heterogeneous in size but similar to intact erythrocytes in phospholipid composition. Although it has been reported that spectrin-free vesicles are released by echinocytogenic agents, pressure-induced vesicles did contain considerable and similar amounts of spectrin irrespective of the difference in size. These results suggest that vesiculation by high pressure is associated with the disruption of the embrane skeleton, as previously seen in pressure-induced hemolysis [Yamaguchi et al. (1989) J. Biochem. 106, 1080-1085].View full abstractDownload PDF (2256K) -
Hideo Ago, Yasuyuki Kitagawa, Akira Fujishima, Yoshiki Matsuura, Yukit ...1991 Volume 110 Issue 3 Pages 360-363
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have determined the crystal structures of two types of human basic fibroblast growth factor, the serine analogue and the wild-type, at 1.6 and 2.5 Å resolution, respectively. Two good heavy atom derivatives were found and used for multiple isomorphous replacement phasing. The atomic coordinates were refined using the Hendrickson & Konnert program for stereochemically restrained refinement against structure factors. The crystallographic R factors were reduced to 15.3% for the serine analogue structure and 16.0% for the wild-type structure. The serine analogue and wild-type structures have been found to be almost identical, the root-mean-square deviation between the corresponding Cα atoms being 0.11Å. Their structures are composed of twelve β-strands forming a barrel and three loops. Their molecules have an approximate threefold internal symmetry and are similar in architecture to that of interleukin-1 β. A possible heparin-binding site, which comprises five basic residues, Lys119, Arg120, Lys125, Lys129, and Lys135, has been revealed by calculating the electrostatic potential energy.View full abstractDownload PDF (1342K) -
Tsukasa Kuroda, Katsuya Mikawa, Hiroyuki Mishima, Akira Kishimoto1991 Volume 110 Issue 3 Pages 364-368
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLimited proteolysis of protein kinase C (PKC) subspecies with Ca2+-dependent neutral protease II (calpain II) was remarkably stimulated by basic polypeptides, such as H1 histone and poly-L-lysine. This stimulatory effect was observed for proteolysis of the active form of PKC, which was associated with phospholipid and diacylglycerol. The inactive form of PKC was far less susceptible to proteolysis, both in the presence and absence of the basic polypeptides. The basic polypeptides did not appear to interact with calpain II, but made the PKC molecule more susceptible to proteolysis. The relative rates of cleavage of type I (γ), H (α), and III (β) PKC were 2:2:1. The available evidence suggests that, like calpain I, calpain II may also contribute to the down-regulation or depletion of PKC.View full abstractDownload PDF (558K) -
Yoshiko Ohno-Iwashita, Machiko Iwamoto, Ken-ichiro Mitsui, Susumu Ando ...1991 Volume 110 Issue 3 Pages 369-375
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have previously suggested the existence of two distinctive states of cholesterol in erythrocyte and lymphoma cell membranes as revealed by high- and low-affinity binding sites for θ-toxin of Clostridium perfringens [Ohno-Iwashita, Y., Iwamoto, M., Mitsui, K., Ando, S., & Nagai, Y. (1988) Eur. J. Biochem. 176, 95-101; Ohno-Iwashita, Y., Iwamoto, M., Ando, S., Mitsui, K., & Iwashita, S. (1990) Biochim. Biophys. Acta 1023, 441-448]. To understand factor(s) which determine membrane cholesterol heterogeneity, we analyzed toxin binding to large unilamellar liposomes composed of cholesterol and phospholipids (phosphatidylcholine/phosphatidylglycerol=82:18, mol/mol). Liposomes containing phospholipids with 18-carbon hydrocarbon chains at both positions 1 and 2 of the glycerol have both high- and low-affinity toxin-binding sites with Kd values similar to those of intact erythrocytes, whereas liposomes with hydrocarbon chains containing 16 or fewer carbons at either position 1 or 2 have only low-affinity toxin-binding sites. The cholesterol/ phospholipid ratio, in addition to the length of phospholipid hydrocarbon chain, also determines the number of toxin-binding sites, indicating that at least these two factors determine the topology of membrane cholesterol by creating distinctively different affinity sites for the toxin. Since θ-toxin binding detects specific populations of membrane cholesterol that are not detectable by the measurements of susceptibility to cholesterol oxidase and cholesterol desorption from membranes, the toxin could provide a unique probe for studying the organization of cholesterol in membranes.View full abstractDownload PDF (1194K) -
Hiroyuki Yana, Kazuo Satake, Yoshio Ueno, Kiyoshi Kondo, Akira Tsugita1991 Volume 110 Issue 3 Pages 376-380
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequence of the α subunit of the allosteric hemerythrin from Lingula unguis was determined. It consists of 117 amino acid residues. Compared with other non-allosteric hemerythrins consisting of identical subunits of 113 amino acid residues, this protein has the deletion of the N-terminal amino acid and the insertion of five amino acids in the same region as in the case of the monomeric myoerythrin from Themiste zostericola. As the amino acid sequence of the β subunit has also been determined [Yano, H., Satake, K., Ueno, Y., & Tsugita, A. Protein Sequence and Data Analysis, in press], the complete sequence analysis of an allosteric hemerythrin has been accomplished for the first time. The difference in the octameric structures of allosteric and non-allosteric hemerythrins are discussed.View full abstractDownload PDF (477K) -
Toshinaga Maeda, Kimikazu Hashing, Rieko Oyama, Koiti Titani, Kiyotosh ...1991 Volume 110 Issue 3 Pages 381-387
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn artificial cell adhesive protein could be engineered by grafting the RGDS tetrapeptide, the core sequence of the major cell adhesive site of fibronectin, to a truncated form of Staphylococcal protein A (tSPA) via cassette mutagenesis of the tSPA expression vector pRIT2T [T. Maeda et al. (1989) J. Biol. Chem. 264, 15165-15168]. We synthesized a panel of tSPA derivatives grafted with various RGDS-containing oligopeptides to address the problem of how the cell adhesive activity of the resulting tSPA derivatives was affected by the length and amino acid sequence of the grafted oligopeptides and by the sites on tSPA where the extra oligopeptides were inserted. The results showed that (i) the amino acid residues flanking the RGDS core sequence played a key role in modulating the cell adhesive activity of the grafted RGDS signal; (ii) at least two sites on tSPA, each corresponding to one of the two HindIII sites of pRIT2T, were competent in sustaining the cell adhesive activity of the grafted signal; and (iii) the divalent tSPA containing the RGDS signal at both sites was more active than monovalent derivatives containing only one signal at either site. These results provide a strategic basis for engineering of artificial cell adhesive proteins by grafting the RGDS signal.View full abstractDownload PDF (854K) -
Koichi Mishima, Masaharu Terashima, Seiji Obara, Kazuo Yamada, Katsuyu ...1991 Volume 110 Issue 3 Pages 388-394
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have reported the purification and characterization of arginine-specific ADP-ribo-syltransferase from hen liver nuclei [Tanigawa, Y. et al. (1984) J. Biol. Chem. 259, 2022-2029] and the DNA-dependent mono(ADP-ribosyl)ation of p33, an acceptor protein in the nuclei [Mishima, K. et al. (1989) Eur. J. Biochem. 179, 267-273]. In the present study, we obtained evidence that among various tissues and cells from chicken, polymorphonuclear cells, so-called heterophils, possess both the ADP-ribosyltransferase and p33 at high levels. Percoll density gradient centrifugation of the postnuclear fraction of the heterophils revealed the co-localization of ADP-ribosyltransferase with p33 in the granule fraction. The enzyme and p33 were purified approximately 219- and 3.77-fold, respectively, from postnuclear pellet fraction to apparent homogeneity. The properties of heterophil ADP-ribosyltransferase and p33 were compared with those of the liver enzyme and p33. The molecular mass of the heterophil enzyme was estimated by SDS-polyacrylamide gel electrophoresis to be 27.5 kDa. The enzyme activity was stimulated by a sulfhydryl agent and inhibited by lysolecithin, NaCl, and inorganic phosphate. The mono(ADP-ribosyl)- ation of p33 was markedly enhanced by polyanion, such as DNA, RNA, or poly(L-gluta-mate). SDS-polyacrylamide gel electrophoretic analysis after limited trypsin proteolysis of p33s, purified from chicken heterophils and liver, showed much the same pattern. Thus, it appears that ADP-ribosyltransferase and p33 present in heterophils are identical to those in the liver, respectively. p33 is considered to be an in situ substrate for ADP-ribosyl-transferase, since it was specifically mono(ADP-ribosyl)ated in permeabilized heterophils. Chicken ADP-ribosyltransferase may be involved in the function of polymorphonuclear cells through modification of the granule protein p33.View full abstractDownload PDF (4541K) -
Yoichi Nakamura, Kinichiro Oda, Susumu Nakada1991 Volume 110 Issue 3 Pages 395-401
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to establish cell lines which complement the growth of temperature-sensitive (ts) mutants of influenza virus, three RNA polymerase and nucleoprotein (NP) genes each linked to the mouse mammary tumor virus LTR were cloned into the bovine papilloma-virus vector DNA. After co-transfection of mouse C127 cells with these recombinant plasmids, a cell line, clone 76, in which the expression of the three polymerase and NP genes could be stimulated by dexamethasone, was established. The clone 76 cells could comple-ment the growth of ts-mutants defective in one of the polymerase subunit genes at the nonpermissive temperature in response to dexamethasone. The results suggest that the simultaneous expression of the three polymerase genes in the same compartment of protein synthesis machinery is required for an efficient complementation of ts-mutant growth.View full abstractDownload PDF (4216K) -
Hidevuki Mukai, Akira Ito, Koii Kishima, Takavoshi Kuno, Chikako Tanak ...1991 Volume 110 Issue 3 Pages 402-406
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe interaction of calmodulin antagonists with a phosphoprotein phosphatase, calcineurin, was investigated using para-nitrophenyl phosphate (pNPP) as a substrate. Calmidazo-lium, a potent calmodulin antagonist, inhibited the Ni2+-stimulated calmodulin- indepen-dent phosphatase activity to much the same extent as it did the Ca2+/calmodulin-stimulated activity. Other calmodulin antagonists, such as trifluoperazine, thioridazine, and W-7, also inhibited the Ni2+-stimulated phosphatase activity. On the other hand, calmidazolium only weakly and partially inhibited the Mn2+- stimulated phosphatase activity and the other calmodulin antagonists examined increased the Ni2+-stimulated activity, in the absence of calmodulin. With the addition of an equimolar amount, as to the inhibited holoenzyme, of the purified B subunit of calcineurin, the Ni2+-stimulated phosphatase activity recovered from 38 to 63% of the control level in the presence of 5μM calmidazolium. When the amount of additional B subunit was increased, the phosphatase activity recovered to 94% of the control level, thereby implying that calmidazolium inhibits the Ni2+-stimulated phosphatase activity by interacting with the B subunit, in the absence of calmodulin. The Mn2+-stimulated phosphatase activity also recovered from the inhibition by calmidazo-lium, but a much larger amount of the B subunit was necessary for the recovery. These results indicate that the Ni2+-and Mn2+-stimulated activities of calcineurin are differentially affected by calmodulin antagonists and that the B subunit plays a crucial role in the expression of the Ni2+-stimulated phosphatase activity.View full abstractDownload PDF (522K) -
Shin-ichi Hayashi, Junko Watanabe, Kei Nakachi, Kaname Kawajiri1991 Volume 110 Issue 3 Pages 407-411
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIndividuals with high genetic risk of lung cancer had previously been identified by Msp polymorphisms of the cytochrome P450IA1 gene. In the present study we analyzed the structures of individual P450IA1 genes by PCR direct sequencing of genomic DNA of each genotype raised by the Msp polymorphisms, which were ascribed to a single point mutation in the 3'-flanking region. We then found a novel point mutation in the coding region of the gene which results in the substitution of Ile for Val at residue 462 in the heme binding region. We further analyzed the genetic association between this amino acid replacement and Msp polymorphisms in the general population, using a new method to detect polymorphisms not recognized by restriction enzymes. The results showed that there are at least two forms of human P450IA1 protein with different primary structures and that one of the forms is closely linked with the lung cancer-susceptible genotype of Msp polymorphisms. Thus Msp polymorphisms, which are associated with increased risk of lung cancer, are linked to at least one amino acid substitution, which gives an important clue, at the molecular level, toward elucidation of increased susceptibility to lung cancer.View full abstractDownload PDF (3939K) -
Isolation and Characterization of a Monosialosylgangliopentaosyl Ceramide from Xenopus laevis OocyteKazuya Hidari, Saki Itonori, Yutaka Sanai, Masako Ohashi, Takeshi Kasa ...1991 Volume 110 Issue 3 Pages 412-416
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA monosialosylgangliopentaosyl ceramide was isolated from Xenopus laevis oocytes. It represented 5.8% of the total acidic glycosphingolipids. From the results of sugarcomposition analysis, enzymatic hydrolysis, permethylation analysis, and negative ion fast atom bombardment mass spectrometry, the structure of the ganglioside was determined to be as follows:
GalNAc β1→4Gal β1→3GalNAc β1→4Gal β1→4Glc β1→1'Cer
NeuAc α2→3
The predominant species of fatty acids were α-hydroxy fatty acids, h22 : 0, h24 : 0, and h24 : 1. The long chain bases of this ganglioside consisted mainly of d18 : 1 sphingosine and phytosphingosine. Other acidic glycolipids were also characterized. The most abundant component of acidic glycolipids was sulfatide, which represented 85.7% of the total acidic glycolipid mixture. GM3, GM2, GMla, and GDla were also detected.View full abstractDownload PDF (3009K) -
Motomu Terasawa, Hiroshi Tokumitsu, Ryoji Kobayashi, Hiroyoshi Hidaka1991 Volume 110 Issue 3 Pages 417-422
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEndogenous phosphorylation of the crude membrane fraction of cultured 3Y1 fibroblast cells was enhanced by the addition of Ca2+/calmodulin. Both Ca2+/calmodulin-dependent protein kinase activity and its substrate were present in a cytoskeletal fraction, obtained as a pellet after washing of the membrane fraction with 2mM EGTA, 0.6 M NaCl, and 1% Triton X-100. The phosphorylatable protein in the Triton X-insoluble fraction was identified by immunoblotting as vimentin. This endogenous phosphorylation induced by calmodulin was inhibited by the addition of KN-62, a specific Ca2+/calmodulin-dependent protein kinase II-inhibitor, in a dose-dependent manner. However, phosphorylation of the 59 kDa protein (vimentin) in this fraction was not stimulated by adding both phosphatidyl serine and cAMP, thereby suggesting the absence of protein kinase C or of cAMP-dependent protein kinase in this fraction. The protein kinase associated with the Triton X-insoluble fraction phosphorylated the Ca2+/calmodulindependent protein kinase II-specific site of synapsin I from the bovine cortex. Two-dimensional phosphopeptide maps of vimentin indicated that a major phosphopeptide phosphorylated by the endogenous calmodulin-dependent kinase also appears to be the same as a major phosphopeptide phosphorylated by the exogenous Ca2+/calmodulin-dependent protein kinase II. Our results suggest that cytoskeleton-associated Ca2+/calmodulin-dependent protein kinase II regulates dynamic cellular functions through the phosphorylation of cytoskeletal elements in non-neural cells.View full abstractDownload PDF (5403K) -
Takashi Kimura, Kaoru Miyazaki, Keisuke Mashima, Jinpei Yamashita, Tak ...1991 Volume 110 Issue 3 Pages 423-428
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt was previously found that rabbit serum contains a growth-inhibitory substance for a tumorigenic rat liver cell line RSV-BRL. In the present study, the growth inhibitor was purified from normal rabbit serum to show a homogeneous protein band with a molecular weight (Mr) of 56 k on SDS-polyacrylamide gel electrophoresis under non-reducing conditions. The purified growth inhibitor, tentatively named rabbit serum-derived growth inhibitor (RSGI), potently inhibited the growth of RSV-BRL and nine kinds of other cell lines including three human tumor cell lines at a concentration of 20ng/ml or higher. The growth-inhibitory effect of RSGI was reversible and appeared to be cytostatic rather than cytotoxic. RSGI was stable to heating at 56°C for 30 min or treatment with 0.1M 2-mercaptoethanol, but labile to heating at 100°C for 3 min or treatment with 1M acetic acid (pH 2.3), 6M urea, 50% (v/v) 1-propanol, or 0.1% (w/v) trypsin. These properties of RSGI suggested that it was different from type β transforming growth factors, tumor necrosis factor-α, and other known growth-regulatory factors.View full abstractDownload PDF (1659K) -
Yasunori Kozutsumi, Takehiro Kawano, Hiroshi Kawasaki, Koichi Suzuki, ...1991 Volume 110 Issue 3 Pages 429-435
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe hydroxylation of CMP-N-acetylneuraminic acid (CMP-NeuAc) in the formation of CMP-N-glycolylneuraminic acid requires several components which comprise an electron transport system. A protein, which replaces one of the components, was purified to homogeneity from a horse erythrocyte lysate. Based on its partial amino acid sequence and immunological cross-reactivity, this protein was identified as soluble cytochrome b5 lacking the membrane domain of microsomal cytochrome b5. The electron transport system involved in CMP-NeuAc hydroxylation was reconstituted, and then characterized using the purified horse soluble cytochrome b5 and a fraction from mouse liver cytosol. The hydroxylation reaction requires a reducing reagent, DTT being the most effective. Either NADH or NADPH was used as an electron donor, but the activity with NADPH amounted to about 74% of that with NADH. The hydroxylation was inhibited by salts and azide due to interruption of the electron transport from NAD(P)H to cytochrome b5 and in the terminal enzyme reaction, respectively.View full abstractDownload PDF (893K) -
Jin-Gu Kim, Jong-Duk Kim1991 Volume 110 Issue 3 Pages 436-442
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSVesicle to micelle transitions of sonicated liposomes of egg yolk phosphatidylcholine (EPC) induced by a homologous series of nonionic surfactants, poly(oxyethylene) cetyl ethers [POE(n) cetyl ether], were investigated by using the method of turbidity titrations. The turbidities of the mixed dispersions of sonicated vesicles and surfactant were systematically measured as a function of the surfactant added for a wide range of lipid concentrations (from 0.51 to 6.35mM EPC). From the titration curves, two threshold points representing onset and complete solubilization of liposomal membranes were determined as a probe for the effect of the length of ethylene oxide (EO) moiety on the phase behavior of ternary system of POE (n) cetyl ethers-EPC-excess water. Patterns of turbidity curves and the surfactant concentrations at two threshold points as well as widths of region between two transitions, where lamellar sheets and mixed micelles may coexist, mainly depended on the length of EO head group. With changing the lengths, solubilization of liposomes and phase diagram showed optimal behavior. That is, in the middle range of EO numbers, it resulted in narrowest coexistence region between onset and complete solubilization. Assuming the equilibrium partitioning model, critical effective molar ratios of surfactant to lipid, Rsat, free surfactant concentrations, Dw, and the partition coefficient of surfactant between bilayer and aqueous phase, K, in surfactant-saturated liposomes were quantitatively determined as a function of EO number. Effective ratios, Rsol, and free surfactant concentration in mixed micelles were also determined. In addition, the effects of CMC and HLB of surfactants on the solubilization of liposome were discussed.View full abstractDownload PDF (840K) -
Shuichi Hiraoka, Kazuki Nukui, Nobuyuki Uetake, Akinori Ohta, Isao Shi ...1991 Volume 110 Issue 3 Pages 443-449
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA simple, specific, and sensitive assay procedure for cardiolipin synthase of Escherichia coli has been developed. This measures the radioactivity of glycerol formed from phosphatidyl[2-3H]glycerol and is mainly based on the findings that 400 mM phosphate and 0.015% Triton X-100 markedly activate the enzyme. Cardiolipin synthase was amplified 760-fold upon induction with isopropyl β-D-thiogalactoside in cells harboring a pBR322 derivative in which the cls gene encoding this enzyme was preceded by the tac promoter. Under these conditions, cardiolipin content increased, membrane potential decreased, spheroplasts became fragile, cells lost viability, and inducer-resistant mutants appeared at a high frequency. The amplification enabled the isolation of an enzyme preparation with a specific activity approximately 10, 000-times higher than that of wild-type whole cell lysate. This purification was simply achieved by extraction of the crude membrane fraction with Triton X-100 and a single phosphocellulose column chromatography. This preparation, together with the crude envelope fraction, was used to characterize the basic properties of E. coli cardiolipin synthase, some of which were utilized in setting up the assay conditions.View full abstractDownload PDF (1958K) -
Masao Nakamura1991 Volume 110 Issue 3 Pages 450-456
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOxygen radical generation in the xanthine- and NADH-oxygen reductase reactions by xanthine oxidase, was demonstrated using the ESR spin trap 5, 5'-dimethy1-1-pyrroline-N-oxide. No xanthine-dependent oxygen radical formation was observed when allopurinol-treated xanthine oxidase was used. The significant superoxide generation in the NADH-oxygen reductase reaction by the enzyme was increased by the addition of menadione and adriamycin. The NADH-menadione and -adriamycin reductase activities of xanthine oxidase were assessed in terms of NADH oxidation. From Lineweaver-Burk plots, the Km and Vmax of xanthine oxidase were estimated to be respectively 51 μM and 5.5 s-1 for menadione and 12 μM and 0.4 s-1 for adriamycin. Allopurinol-inactivated xanthine oxi-dase generates superoxide and OH. radicals in the presence of NADH and menadione or adriamycin to the same extent as the native enzyme. Adriamycin radicals were observed when the reactions were carried out under an atmosphere of argon. The effects of superoxide dismutase and catalase revealed that OH. radicals were mainly generated through the direct reaction of H2O2 with semiquinoid forms of menadione and adriamycin.View full abstractDownload PDF (691K) -
Tamotsu Hoshino, Akihiro Mizutani, Shoichi Shimizu, Hiroyoshi Hidaka, ...1991 Volume 110 Issue 3 Pages 457-461
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe lipase production of a plant pathogenic fungus, Fusarium oxysporum f. sp. lini SUF 402, was induced by fat as the carbon source, and its release was stimulated by the infusion of intracellular free calcium ion with a calcium ionophore, A23187. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7, a calmodulin inhibitor) and 1-[N, O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62, a Ca2+/calmodulin dependent protein kinase II inhibitor) reduced the extracellular release of lipase in vivo. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7, a protein kinase C inhibitor) did not have this ability. After K2H32PO4, had been incorporated into the cells, they were treated with W-7 or KN-62 and stimulated by Ca2+ ionophore. On SDS-PAGE of intracellular proteins followed by autoradiography, W-7- and KN-62-treated cells showed inhibition of the incorporation of 32Pi into the 20 kDa protein resulting from Ca2+ stimulation. F. oxysporum had calmodulin (CaM)-dependent protein kinase activity in the cytoplasmic fraction and had the ability to phosphorylate of syntide 2, a specific substrate of CaM kinase II. The partially purified CaM-dependent protein kinase was inhibited by 10 μM KN-62 in vitro. Increase of the intracellular Ca2+ concentration of F. oxysporum activated CaM and CaM-dependent protein kinase, resulting in the extracellular lipase release. These results suggest the existence of a Ca2+ signalling system in F. oxysporum like those observed in higher eucaryotes.View full abstractDownload PDF (1511K) -
Lois L. Hoyer, Peter Roggentin, Roland Schauer, Eric R. Vimr1991 Volume 110 Issue 3 Pages 462-467
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSubclones containing the Salmonella typhimurium LT2 sialidase gene, nanH, were expressed in Escherichia coli from multicopy derivatives of pBR329. The cloned sialidase structural gene directed overproduction of sialidase polypeptide which was detected as the major soluble protein species in cell-free extracts. Overproduced enzyme was purified to near electrophoretic homogeneity after 65-fold enrichment using conventional preparative techniques. Unlike all previously investigated sialidases, S. typhimurium sialidase was positively charged (pI≥9.0). Km, Vmax, and turnover number of the purified sialidase, measured using 2'-(4-methylumbellifery1)-α-D-N-acetylneuraminic acid (MUNeu5Ac), were 0.25 mM, 5, 200 nmol min-1 and 2, 700 s-1, respectively. These values are the highest yet reported for a sialidase. Sialidase was inhibited by 2-deoxy-2, 3-didehydro-N-acetyl-neuraminic acid at unusually high concentrations (K1=0.38 mM), but not by 20 mM N-acetylneuraminic acid. Divalent cations were not required for activity. The pH optimum for hydrolysis of MUNeu5Ac was between 5.5 and 7.0 and depended on the assay buffer system. Substrate specificity measurements using natural sialoglycoconjugates showed a 260-fold kinetic preference for sialyl α2→3 linkages when compared with α2→6 bound sialic acids. The enzyme also efficiently cleaved residues from glycoproteins and ganglio-sides, but not from mucin or sialohomopolysaccharides. S. typhimurium sialidase is thus the first bacterial enzyme to be described with influenza A virus sialidase-like kinetic preference for sialyl α2→3 linkages and to have a basic pI.View full abstractDownload PDF (3080K)
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